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Title: Investigating the programming of Type I highly reducing iterative polyketide synthases
Author: Roberts , Douglas
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2014
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Previous work, reported in the literature, has suggested that isolated catalytic domains from highly reducing iterative polyketide synthases (HR iPKS) should be able to perform their programmed function independently of the complete polyketide synthase (PKS). To test this hypothesis, the isolated enoyl reductase domain (ER) of squalestatin tetraketide synthase (SQTKS) was produced and tested in vitro with substrate mimics of intermediates of the biosynthesis of squalestatin tetraketide 54. Both the selectivity of the enzyme and the stereocontrol of the catalyzed reduction were investigated. Two assay methods were designed using UV absorption and LCMS to measure the initial rate and percentage turnover of the enzymatic reaction to test the substrate selectivity of the ER domain. The results of these assays demonstrated that substrate recognition was dependent on methylation of the substrate and in particular methylation at the 8-position was critical for preventing the enzymatic reduction from occurring. A second set of assays was designed to test the stereochemical outcome of the enzymatic reduction. These results demonstrate that although the transfer of ~-hydride from NADPH to the substrate is controlled, the isolated ER domain is unable to control the astereocentre. The results from this study have shown that the ER domain is able to recognize substrates without the complete PKS but for the reduction to be stereo controlled the complete PKS may be required. To better understand the results a computer model of the isolated ER domain was created and by examining the model four amino acids (F 1941, I 2001 , L 2146 and I 2147) that could control substrate recognition in the ER domain were highlighted. Also three other amino acids (R 1924, D 1925 and M 1927) that might be involved in controlling the stereochemistry of the a-position were also found in the active site of the ER domain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available